While over 500,000 surgeries correcting bone deformities and critical size defects occur each year in the US, 50% of the procedures end in failure. Therefore, using molecular engineering approaches to regenerate lost bone with normal anatomic contour, physical properties and biological function is of enormous importance. MicroRNAs (miRNAs) are non-coding RNAs that are emerging as new and powerful drug targets. Currently there are 157 preclinical and clinical trials of miRNA-based therapeutics. We have identified a novel miRNA, miR-335-5p, which targets the 3'-UTR sequence of DKK1, a Wnt signal antagonist, and down-regulates DKK1 expression. We also revealed that miR-335-5p is an important regulator for osteogenic differentiation. We have further generated a transgenic mouse line in which miR-335-5p gene is overexpressed in bone tissue showing enhanced expression of marker genes and increased bone mass accrual. Based on these interesting findings we plan to use miR-335-5p as a therapeutic target to enhance oral and craniofacial bone regeneration using our newly synthesized and characterized lipidoid-miRNA nanoparticles as a novel delivery vehicle. Aim 1. We will determine regulatory and osteogenic effects of miR-335-5p overexpression in bone regeneration and wound repairing using a transgenic animal model. Our hypothesis is that since miR-335-5p transgene is incorporated into mouse genome its super-physical transcript levels will boost the bone forming capacity in target cells. Aim 2. We will develop two therapeutic strategies with a nanoparticle lipidoid system to deliver miR-335-5p molecules in vivo for oral and craniofacial bone regeneration. Firstly, we will transplant miR-335-5p-modified bone marrow stromal cells into bone wound site to enhance bone formation. Our hypothesis is that miR-335-5p specifically suppresses DKK1 and activates Wnt/??Catenin signals, which further triggers an osteogenic cascade leading BMSC differentiation towards osteoblastic lineage and an increased bone formation capability. Secondly, we will determine the effect of miR-335-5p in promoting bone-forming cells in situ and enhancing wound healing and tissue regeneration. Our hypothesis is that local administration of miR-335-5p in wound site directly leads mesenchymal stem cells differentiation into bone forming cells, accelerating wound healing and bone regeneration. This translational research will explore the potential of miR-335-5p as a therapeutic agent for promoting bone regeneration and wound healing. This innovative miRNA-based therapy has potential for the clinical treatment of other bone disorders including osteoporosis, fracture, periodontitis and impaired osseointegration of bone/joint implants.